Treatment of neurological disorders by dsRNA administration

ABSTRACT

The present invention relates to methods to treat neurological disorders comprising intrathecal injection of an effective amount of a double-stranded (ds) RNA into a subject in need, wherein the dsRNA inhibits the expression of a target gene and to pharmaceutical compositions useful for such treatment.

This application claims benefit of priority from Provisional ApplicationNo. 60/408,000 filed on Sep. 4, 2002, Provisional Application No.60/457,971 filed on Mar. 27, 2003, International Application No.PCT/EP2003/009787, filed on Sep. 3, 2003, and Utility patent applicationSer. No. 10/525,312 filed on Mar. 24, 2005, now abandoned, all of whichare incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods of treatment neurologicaldisorders and to a pharmaceutical composition for the treatment ofchronic pain.

BACKGROUND OF THE INVENTION

Methods of inhibiting the expression of genes through shortsingle-stranded oligonucleotides or oligoribonucleotides or modifiedoligonucleotides perfectly complementary to the target mRNA are known as“antisense”. The use of antisense oligonucleotides (ASOs) as a tool tohelp elucidate gene function is well-described. Antisenseoligonucleotides are also being evaluated as medicaments for a widevariety of diseases.

As an alternative to antisense, sequence-specific degradation of mRNAswith oligonucleotides can also be triggered by short RNA duplexes by anRNA interference (RNAi) mechanism. RNA interference is a process ofsequence-specific, post-transcriptional gene silencing initiated bydouble-stranded RNA that is homologous in sequence to the silenced gene.The modulation of the function of a target nucleic acid byoligoribonucleotides which inhibit the expression of said target nucleicacid is generally referred to as “RNAi” or “RNA interference”. Effectivetarget-gene specific inhibition is usually achieved by shortdouble-stranded (ds) oligoribonucleotide and with an overhang ofapproximately 2 nucleotides at the ends of at least 1 strand of theduplex. Such double-stranded oligoribonucleotides are known as shortinterfering RNAs (siRNAs) and have for instance been used as a tools tohelp elucidate gene function.

Great efforts are being made to develop oligonucleotides inhibiting theexpression of specific target gene for therapeutic uses. One of theproblems encountered is that, due to the special characteristics ofoligonucleotides (such as for example high molecular weight, highamounts of negative charge, metabolic instability), delivery of freeoligonucleotides to target tissues is generally much more limited interms of the variety of disease target tissues, than for small moleculeinhibitors: for instance, free oligonucleotides have low bioavailabilitywhen given orally to patients, systemic delivery of oligonucleotidesleads to high levels of drug concentrated in a small number of organs,for example in liver, spleen and kidney, where the distribution isdependent on the format of the oligonucleotide (Feng et al., in 2000,European Journal of Pharmaceutical Sciences 10, 179-186). Delivery ofoligonucleotides to the Central Nervous System (CNS) poses particularproblems due to the blood brain barrier (BBB) that free oligonucleotidescannot cross. One means to deliver oligonucleotides into the CNS isintrathecal delivery. However, the oligonucleotides need also to beefficiently internalised into target cells of the CNS in order toachieve the desired therapeutic effect. Usually, delivery reagents suchas liposomes, cationic lipids, nanoparticles forming complexes areutilized in order to aid the intracellular internalization ofoligonucleotides into cells of neuronal origin. However, it is ofconsiderable economic and technical advantage in the development ofdrugs if the desired pharmacological effects can be achieved without theuse of tissue delivery reagents. So far, the only report describingshort dsRNAs entering mammalian cells without the aid of a deliveryreagent show a poor effect (Milhaud, Pierre G. et al., J. InterferonRes. (1991), 11(5), 261-5). We have now surprisingly found in accordancewith the present invention, that intrathecally delivered siRNAsefficiently enter CNS tissues and are efficiently internalized intocells of the CNS system. Thus, the present invention now provides forthe first time a method for functional downregulation of target genes bydsRNA in the CNS in vivo, thereby affecting the disease phenotype, bydelivering siRNA to the CNS.

SUMMARY OF THE INVENTION

The present invention relates to a method to treat or ameliorateneurological disorders comprising intrathecal injection of an effectiveamount of a double stranded (ds) RNA into a subject in need, whereinsaid dsRNA inhibits the expression of a target gene. In a preferredembodiment the neurological disorder is selected from the groupconsisting of Alzheimer, Parkinson, multiple sclerosis, schizophrenia,epilepsy, depression and pain. In a more preferred embodiment, thedisorder is chronic pain, preferably chronic neuropathic pain, cancerpain or osteoarthritis pain. In another preferred embodiment, thedisorder is allodynia or hyperalgesia. Alternatively, the disorder isinflammatory chronic pain. In another preferred embodiment, the targetgene is selected from the group consisting of purine receptors P1 or P2,Galanin R1 receptor, Vanilloid receptors 1, voltage gated calciumchannel (N-type), the tetrodotoxin-resistant sodium channel Nav1.8(PN3/SNS), TRPM8, IL-24, IL-20Ralpha or IL-20Rbeta. Particularlypreferred are the P2 receptors, most preferred is P₂X₃ or P₂X₂. Furtherpreferred target genes include Mob-5 or MMP7.

The subject in need is preferably mammalian. In one aspect of thisinvention the subject in need is rodent, preferably a rat. In a relatedaspect the subject in need is a monkey or a human.

In accordance with one aspect of the present invention, the amount ofdsRNA that is intrathecally injected is 50 μg to 1500 μg, preferablymore than 180 μg, more preferably more than 200 μg, more than 300 μg ormore than 400 μg.

In another aspect of the present invention, the dsRNA comprises adouble-stranded region of 15 to 25 nt, preferably of 19 nt. In a relatedaspect, the dsRNA comprises a 3′ overhang on the antisense or the sensestrand or both strands of at least one nucleotide, preferably 1, 2, 3 or4 nucleotides. In a preferred embodiment, the penultimate nucleotide ofthe overhang is complementary to the mRNA target strand. In anotherpreferred embodiment, the overhang contains at least one modifiednucleotide, a preferred modification is a 2-MOE modification. In afurther preferred embodiment, the overhang comprises at least one UUand/or dTdT group. Also preferred is an overhang comprising UUUU orconsisting of UUUU. In yet a further embodiment, the dsRNA comprises atleast one modified linkage, preferred is at least one phosphorothioatelinkage.

Another aspect of the present invention relates to the use of dsRNA forthe treatment of chronic pain. The dsRNA is preferably administered byintrathecal injection to a subject in need and inhibits the expressionof a target gene. In a preferred embodiment the chronic pain is chronicneuropathic pain, in another preferred embodiment the chronic pain isselected from the group consisting of cancer pain, osteoarthritis pain,allodynia or hyperalgesia. In further preferred embodiment the targetedgene is a gene encoding a purine receptors P1 or P2, Galanin R1receptor, Vanilloid receptors 1, voltage gated calcium channel (N-type),the tetrodotoxin-resistant sodium channel Nav1.8 (PN3/SNS), TRPM8,IL-24, IL-20Ralpha or IL-20Rbeta, most preferred is a gene encoding P₂X₃or P₂X₂. Further preferred genes include Mob-5 or MMP7.

Another aspect of the present invention relates to pharmaceuticalcompositions comprising an effective amount of dsRNA, wherein the dsRNAinhibits the expression of a target gene. These target genes arepreferably overexpressed in chronic pain, preferably chronic neuropathicpain. Preferred target genes are the purine receptors P1 or P2, GalaninR1 receptor, Vanilloid receptors 1, voltage gated calcium channel(N-type), the tetrodotoxin-resistant sodium channel Nav1.8 (PN3/SNS),TRPM8, IL-24, IL-20Ralpha or IL-20Rbeta. Particularly preferred are theP2 receptors, most preferred is P₂X₃ or P₂X₂. Further preferred targetgenes include Mob-5 or MMP7. In another preferred embodiment, thepharmaceutical composition comprising an effective amount of a doublestranded RNA is selected from the group consisting of SEQ ID Nos: 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15 and 16.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill from the following description.It should be understood, however, that the following description and thespecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only. Various changes andmodifications within the spirit and scope of the disclosed inventionwill become readily apparent to those skilled in the art from readingthe following description and from reading the other parts of thepresent disclosure.

DETAILED DESCRIPTION OF THE INVENTION

It is contemplated that the invention described herein is not limited tothe particular methodology, protocols, and reagents described as thesemay vary. It is also to be understood that the terminology used hereinis for the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention in any way.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices and materials are now described. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing the materials and methodologies that are reported in thepublication which might be used in connection with the invention.

In practicing the present invention, many conventional techniques inmolecular biology are used. These techniques are well known and areexplained in, for example, Harlow, E. and Lane, eds., 1988, “Antibodies:A Laboratory Manual”, Cold Spring Harbor Press, Cold Spring Harbor,Current Protocols in Molecular Biology, Volumes I, II, and III, 1997 (F.M. Ausubel ed.); Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II, 1985(D. N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.);Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription andTranslation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986(R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press);Perbal, 1984, A Practical Guide to Molecular Cloning; the series,Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors forMammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold SpringHarbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wuand Grossman, and Wu, eds., respectively).

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise.

As used herein, “double-stranded ribonucleic acid (dsRNA)”, as usedherein, refers to an oligoribonucleotide or polyribonucleotide, modifiedor unmodified, and fragments or portions thereof, of genomic orsynthetic origin or derived from the expression of a vector, which maybe partly or fully double-stranded and which may be blunt-ended orcontain a 5′- and/or 3′-overhang, and also may be of a hairpin formcomprising a single oligoribonucleotide which folds back upon itself togive a double-stranded region.

As used herein “siRNA” denotes short interfering RNAs and refers toshort double stranded ribonucleic acids useful for RNAi.

As used herein “inhibition” of gene expression means the reduction ofthe expression of said gene by at least 10%, 33%, 50%, 90%, 95% or 99%.

As used herein, the term “form” of or “format” of in relation tooligonucleotides refers to different chemical nature of theoligoribonucleotide, in particular to modifications as compared tonaturally occurring ribonucleotides, such as for instance chemicallymodified 2′OH groups of the ribose moiety or the modifiedinternucleosidic linkages such as phosphothioate linkages, or themodified nucleobases such as for example 5-methyl-C.

As used herein “Subject” refers to any human or nonhuman organism.Preferred are mammalian organisms.

As used herein the term nucleotide means ribonucleotide ordeoxyribonucleotide, the terms oligonucleotide and oligoribonucleotideare interchangeable and refer, depending on the context, to modified orunmodified oligonucleotides comprising ribonucleotides and/ordeoxyribonucleotides.

The present invention is based on the surprising discovery thatintrathecally injected dsRNA inhibited the expression of a target genethereby leading to a therapeutic effect in vivo, thus providing for thefirst time that a successful therapeutic treatment of a neurologicaldisorder has been achieved by administration of dsRNA. Furthermore, themagnitude of the pharmacological effect from the siRNA on allodynia ismuch greater than that from the analogous antisense oligonucleotide. Forinstance, a dose limiting toxicity from use of the antisenseoligonucleotide does not allow a pharmacological effect on allodynia tobe observed, whereas no such does limiting toxicity was observed fromuse of the siRNA, showing the possible advantages of using siRNAs overASOs. The present invention makes therefore dsRNA available for thetherapeutic treatment of neurological diseases.

In accordance with the present invention, the ribonucleic acid used forinhibition will have at least a partially double-stranded character, butmay also be totally double-stranded. The RNA can be a single strand thatis self-complementary or may comprise two or more separate complementarystrands.

Particularly preferred in accordance with the present invention areshort double-stranded RNAs, also termed siRNAs, having a length of 10 to50 nucleotides, preferably 15 to 25 nucleotides. Yet more preferred aredsRNA's composed of oligoribonucleotides having a duplex length of 17 to21 ribonucleotides. Even more preferred are oligoribonucleotides havinga duplex length of 19 ribonucleotides.

The efficiency, i.e. the degree of inhibition of the target gene, isdependent on a number of different factors including the specificity ofthe dsRNA for its target sequence. In this context, specificity meanshomology, i.e. sequence identity between the dsRNA in the duplex regionand the target sequence. It is understood by a person skilled in the artthat 100% sequence identity is not required in order to achievesignificant inhibition. Normally, at least 75% sequence identity betweenthe dsRNA and the target sequence is sufficient in order to inhibitexpression of the target nucleic acid. Preferred is a sequence identityof at least 80%, more preferred is a sequence identity of at least 90%.Most preferred is a sequence identity of at least 95% between the dsRNAand the target sequence. The best is clearly 100%. In order to targetonly the desired target mRNA, the siRNA reagent should have 100%homology to the target mRNA and at least 2 mismatched nucleotides to allother genes present in the cell or organism. Methods to analyze andidentify ds RNAs with sufficient sequence identity in order toeffectively inhibit expression of a specific target sequence are knownin the art. Sequence identity may be optimized by sequence comparisonand alignment algorithms known in the art (see Gribskov and Devereux,Sequence Analysis Primer, Stockton Press, 1991, and references citedtherein) and calculating the percent difference between the nucleotidesequences by, for example, the Smith-Waterman algorithm as implementedin the BESTFIT software program using default parameters (e.g.,University of Wisconsin Genetic Computing Group). Another factoraffecting the efficiency of the RNAi reagent is the target region of thetarget mRNA. The region of a target mRNA effective for inhibition by theRNAi reagent may be determined by experimentation. Most preferred mRNAtarget region would be the coding region. Also preferred areuntranslated regions, particularly the 3′-UTR, splice junctions. Forinstance, transfection assays as described in Elbashir et al. (2001) maybe performed for this purpose. A number of other suitable assays andmethods exist in the art which are well known to a person skilled in theart.

The dsRNA according to the present invention may also contain modifiednucleotide residues. As anyone having skill in the art of drugdevelopment would readily understand, siRNAs can exist in variousformats as described in Tolen et al. 2002, Nucl. Acids Res. 30,1757-1766; Elbashir S. M. et al, 2001 EMBO J., 20, 6877-6888; FEBS 2002,521, 195-199; Current Biology 2001, 11, 1776-1780; Nature Biotech. 2002,19, 497-500; Nature Biotech. 2002, 19, 505-508; Nucleic Acids Research2002, 20, 1757-1766; Science 2002, 296, 5567, 550-553; Methods (SanDiego, Calif., United States) 2002, 26(2), 199-213.

The dsRNA may be blunt ended or ligated at or on at least one end witheither loops composed of ribonucleotides or deoxyribonucleotides or achemical synthetic linker (WO00/44895). In a preferred embodiment, theribonucleic acid contains 3′-end nucleotide overhangs on the antisensestrand and/or the sense strands of the dsRNA of at least oneribonucleotide or deoxyribonucleotide, or modified nucleotide. Preferredare overhangs with 1, 2, 3 or 4 nucleotides. The overhangs may containboth ribonucleotide(s) and deoxyribonucleotide(s) which in addition maycontain modified sugar moieties. The overhang may be of any sequence,but in a preferred embodiment, the overhang is complementary to thetarget mRNA strand. In another preferred embodiment the overhangcontains at least one UU group or dTdT group. In another preferredembodiment, the overhang on the antisense strand has the penultimateoverhanging nucleotide complementary to the mRNA target strand.Preferably, such an overhang is a 2-nucleotides overhang. In a furtherpreferred embodiment, the overhang is composed of 4 Us.

In another preferred embodiment, the extreme 3′-position of the siRNA isa hydroxyl group. Additionally, the 5′-end may be a hydroxyl orphosphate group.

The sugar moieties may be unmodified or modified. Preferred modifiedsugar moieties oligonucleotides comprise one of the following at the 2′position: F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- orN-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C 1 to C 10 alkyl or C2 to C10alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3,O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nNR2, O(CH2)nCH3, O(CH2)nONH2, andO(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Otherpreferred oligonucleotides comprise one of the following at the 2′position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, Cl, Br, CN, CF3, OCF3, SOCH3,SO2 CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy (2′-O—CH2 CH2 OCH3, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78, 486-504) i.e., an alkoxyalkoxy group. A further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, 2′-methoxy (2′-O—CH3),2′-aminopropoxy (2′-OCH2 CH2 CH2 NH2). A further preferred modificationof this category is the bicyclic class of modifications knowncollectively as LNAs (Locked Nucleic Acids) as described in Rajwanshi etal., Angew. Chem. Int. Ed. 2000, 39, 1656-1659. One of skill in the artmay use conventional methods to created such modified sugar structures.Representative United States patents that teach the preparation of suchmodified sugar structures include, but are not limited to, U.S. Pat.Nos. 4,981,957; 5,118,800; 5,700,920 and 5,969,116 each of which isincorporated by reference herein in its entirety.

The internucleoside linkage of the dsRNA may be the “normal” 3′ to 5′phosphodiester linkage or contain at least one chemically modifiedlinkage. In a preferred embodiment, the at least one overhangingnucleotides contains one or more modified linkages, whereas thedouble-stranded part of the oligonucleotide contains phosphodiesterinternucleoside linkages. Preferred modified linkages include but arenot limited to, for example, those disclosed in U.S. Pat. Nos.3,687,808; 4,469,863 and 5,625,050; each of which is incorporated byreference herein in its entirety. In a preferred embodiment the linkagesare phosphorothioates, chiral phosphorothioates or phosphorodithioates.Techniques for the synthesis of compounds containing oligonucleotideswith modified linkages as described above may be achieved usingconventional methodologies, and are familiar to one of skill in the art.

The oligoribonucleotides may be prepared by chemical synthesis (MicuraR., Angewandte Chemie, International Edition (2002), 41(13), 2265-2268)on commercially available or homemade oligonucleotide synthesizers usinga number of different chemistries that are well known in the art. Theoligonucleotide may also be prepared by in vitro transcription of asuitable template using for instance a commercially available kit suchas the Silencer™ siRNA construction kit by Ambion. Alternatively, theoligoribonucleotides may be synthesized by transcription of siRNA'sintracellularly from plasmids through both transient or stabletransfection (Paddison P J et al., 2002, Genes and Development 16,948-958, Paul et al., 2002, Nat. Biotech 29, 505-508).

The effect of dsRNA on gene expression will typically result inexpression of the target gene being inhibited by at least 10%, 33%, 50%,90%, 95% or 99% when compared to a cell not treated according to thepresent invention. Lower doses of administered material, lowerconcentrations of dsRNA in the cell and/or longer times afteradministration of dsRNA may result in inhibition at a lower level and/orin a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%,or 95% of targeted cells). However, it is within the skill of the art toadapt conditions to provide the desired result. Quantitation of geneexpression can be established by assessing the amount of the targetedgene product in the cell. For example, any mRNA transcribed from thetarget gene may be detected with a hybridization probe, or RT-PCR basedmethodologies, or translated polypeptide may be detected with anantibody raised against the encoded polypeptide.

The dsRNA is delivered in accordance with the present application byintrathecal injection (i.e. injection into the spinal fluid which bathesthe brain and spinal chord tissue). Intrathecal injection of siRNA'sinto the spinal fluid can be performed as a bolus injection or viaminipumps which can be implanted beneath the skin, providing a regularand constant delivery of siRNA into the spinal fluid. The circulation ofthe spinal fluid from the choroid plexus, where it is produced, downaround the spinal chord and dorsal root ganglia and subsequently up pastthe cerebellum and over the cortex to the arachnoid granulations, wherethe fluid can exit the CNS, that, depending upon size, stability, andsolubility of the compounds injected, molecules delivered intrathecallycould hit targets throughout the entire CNS.

The amount of intrathecally injected dsRNA may vary from one target geneto another target gene and the appropriate amount that has to be appliedmay have to be determined individually for each target gene. Typicallythis amount will be in the range between 10 μg to 2 mg, preferably 50 μgto 1500 μg, more preferably 100 μg to 1000 μg. It has been found inaccordance with the present invention that the dose limiting toxicitywas at much lower amount of oligoribonucleotides for ASOs than forsiRNA. For instance, whereas a dose limiting toxicity in the rat modelwas observed at 180 μg per day for ASOs, such a dose limiting toxicitywas still absent at 400 μg per day for siRNAs. Thus, in a preferredembodiment of the present invention the dose of the intrathecallyinjected slRNA is at least 50 μg, more preferably at least 100 μg, morepreferably at least 150 μg per day, more preferably at least 180 μg,more preferably at least 200 μg, more preferably at least 300 μg, mostpreferably at least 400 μg. It will be apparent to a person of skill inthe art that the dose of intrathecally injected dsRNA will have to beadjusted appropriately in other organisms, the appropriate dose forhumans, for instance, may be considerably higher.

In accordance with the present invention, the gene to be inhibited isexpressed in the CNS. The target gene is often, but not always a genewhich is misregulated, often upregulated in a given disease state.Examples for genes expressed in the CNS are genes for cytokines, genescausal for neuro-degeneration or regeneration such as Alzheimer orParkinson or multiple sclerosis, viral genes from viruses infecting theCNS, genes causal for schizophrenia, epilepsy or depression. In apreferred embodiment the gene is causal for pain.

Pain is a term that encompasses a spectrum of clinical states. Acutepain serves as a physiological warning for a potentially tissue-damagingsituation. Chronic pain occurs when the stimulus and pain are unrelatedand the pain is no longer a protective mechanism. Chronic pain statesare characterised by a number of clinical features. As well asspontaneous pain, patients may exhibit hyperalgesia (a greatlyexaggerated response to a noxious mechanical, temperature or chemical),and allodynia (previously non-noxious stimuli are now perceived aspainful). All these features result from a complex series of eventsinvolving changes in the function of sensory nerves in the periphery andin the processing of sensory information in the spinal cord and brain.These changes occur in response to direct neuronal damage or in responseto mediators released during tissue damage or inflammation. Broadlyspeaking, chronic pain syndromes can be defined as inflammatory (alsoknown as nociceptive) or neuropathic. Chronic inflammatory pain, as itsname suggests, occurs during conditions in which there is underlyinginflammation such as rheumatoid arthritis, burns, muscle damage orsurgical wounds. Knowledge of the mechanisms underlying inflammatorypain has advanced considerably over recent years and it is known toinvolve a variety of mediators and their activation and sensitization ofthe peripheral terminals of sensory nerves and the consequent longerterm changes in reactivity of spinal cord neurons. Chronic neuropathicpain is caused where there is a primary lesion or dysfunction of thenervous system and occurs, for example, during conditions such astrigeminal neuralgia, diabetic neuropathy, post-herpetic neuralgia,amputation or physical nerve damage. Chronic neuropathic pain resultsfrom damage to nerves by trauma, by diseases such as diabetes, herpeszoster, or late-stage cancer (see below), or by chemical injury (e.g.some anti-HIV drugs). It may also develop after amputation (includingmastectomy), and is involved in some low-back pain. The mechanisms ofchronic neuropathic pain are poorly understood but are thought toinvolve spontaneous firing of sensory nerves due to the novel expressionof certain classes of ion channel, sprouting of sensory fibres intodifferent layers of the spinal cord, and changes in the expression ofvarious neurotransmitters and receptors in the sensory nerves and spinalcord. Traditionally chronic neuropathic pain has proven to beintractable and is resistant to the standard non-steroidal and opiateanalgesics. There is therefore clearly an unmet clinical need for newanalgesics to treat this type of pain. Cancer pain is the most commonchronic pain syndrome (with probably inflammatory and neuropathiccomponents). It is estimated that one third of patients with advancedcancer will develop skeletal metastases, particularly in breast,prostate and lung cancer. Metastatic bone disease commonly results inbone pain that is usually located to a discrete area and is described asa deep, boring sensation that aches and burns, accompanied by episodesof stabbing discomfort. The mechanisms responsible for bone cancer painare unknown but it probably involves structural damage, periostealirritation and nerve entrapment. There is evidence for the disruption ofnormal bone metabolism and the production of inflammatory prostaglandinsand cytokines. Current treatment of bone cancer pain rests with opiatesbut the doses required results in unacceptable side-effects and at least20% of patients still have uncontrolled pain. Novel, well tolerated andeffective analgesics are desired to optimise the quality of life ofthese patients (Coleman R E (1997) Cancer 80; 1588-1594). Osteoarthritispain is the most common form of chronic neuropathic pain (with probablyinflammatory and neuropathic components) for which people visit generalpractitioners. Osteoarthritis is a chronic disease involving progressivestructural changes in joint tissues, principally cartilage, synovium andsubchondral bone. Typically, arthritic joints exhibit cartilage oedemaand erosion, subchondral bone and synovial thickening, and formation ofbony oesteophytes, all contributing to a deformation of the articularsurface. The principal clinical symptom of osteoarthritis is pain,although the mechanisms underlying the chronic neuropathic pain in thiscondition are not understood.

Thus in another embodiment of the present invention, the gene is causalfor chronic pain. Genes causal for pain can be determined using forinstance the animal models described hereinbelow. Furthermore, a varietyof genes are known to be implicated with chronic pain. For example genesencoding a member of the Vanilloid receptors 1 (NM_(—)080706,NM_(—)080705, NM_(—)080705, NM_(—)080704), or a voltage gated calciumchannel (N-type), especially the alpha2 delta1 subunit a2d1(NM_(—)000722) and the a1B subunit (M94172 and M94173), or metabotropicglutamate receptor 1 (mGluR1) (Fundytus M. E. et al., British Journal ofPharmacology (2001), 132(1), 354-367) or the tetrodotoxin-resistantsodium channel Nav1.8 (PN3/SNS) (Yoshimura N. et al., Journal ofNeuroscience (2001), 21(21), 8690-8696).

In a preferred embodiment of the present invention, the gene encodes amember of the family of the purine receptors P₁ or P₂ (Ralevic &Burnstock, Pharmacological Reviews 50 (1998), 413-492), preferably amember of the P₂Y or P₂X subclass. More preferred are the P₂X₃ or theP₂X₂ gene. Other examples of preferred genes include, but are notlimited to Cathepsin S (NM_(—)004079), TrpM8 (NM_(—)024080), the GalaninR1 receptor (Jacoby A S et al., Genomics (1997) 45:3 496-508,NM_(—)012958, NM_(—)008082, NM_(—)001480) or the genes described in U.S.Patent Application 60/369,893 such as for instance IL-24 (NM_(—)006850),IL-20Ralpha or IL-20Rbeta (NM_(—)014432 and AAZ20504).

In another preferred embodiment the gene encodes a member of the Mob-5family. The term “Mob-5” as used herein refers to Mob-5, Genbank #AAF75553 as well as the human ortholog of this protein, Interleukin 24(Genbank # AAA91780). The human ortholog of rat Mob-5 is also known ashMDA-7 as well as “suppression of tumorigenicity 16 (Jiang, H et al.,Oncogene 11, 2477-2486, 1995). Included in the definition of the abovegenes are any and all forms of these polypeptides including, but notlimited to, variants, partial forms, isoforms, precursor forms, fulllength polypeptides, fusion proteins or fragments of any of the above,from human or any other species. Apparent variants of Mob-5 include forinstance c49a Genbank, Accession Number NM; AAB69171. Homologs of theabove genes, which would be apparent to one of skill in the art, arealso meant to be included in this definition. It is also contemplatedthat the term refers to the above genes isolated from naturallyoccurring sources of any species such as genomic DNA libraries as wellas genetically engineered host cells comprising expression systems, orproduced by chemical synthesis using, for instance, automated peptidesynthesizers or a combination of such methods. Means for isolating andpreparing such polypeptides are well understood in the art.

In another preferred embodiment the gene encodes MMP7 (matrilysin), amatrix metalloproteinase. The term “MMP7” refers to any and all forms ofthis polypeptide including, but not limited to, variants, partial forms,isoforms, precursor forms, the full length polypeptide, fusion proteinscontaining the MMP7 sequence or fragments of any of the above, fromhuman or any other species. The sequence of rat MMP7 may be found inGenbank, Accession Number NM_(—)012864. Homologs and orthologs of MMP7,which would be apparent to one of skill in the art, are meant to beincluded in this definition. It is also contemplated that the termrefers to MMP7 isolated from naturally occurring sources of any speciessuch as genomic DNA libraries as well as genetically engineered hostcells comprising expression systems, or produced by chemical synthesisusing, for instance, automated peptide synthesizers or a combination ofsuch methods. Means for isolating and preparing such polypeptides arewell understood in the art.

In a preferred embodiment the gene that is targeted is of mammalianorigin, in a more preferred embodiment the gene is a rodent gene, mostpreferred is a rat gene. In another preferred embodiment the gene is amonkey or a human gene.

Another aspect of the present invention provides a pharmaceuticalcomposition comprising an effective amount of a double stranded RNAinhibiting the expression of a gene causal for pain in an amounteffective to treat chronic pain in a subject in need. In a preferredembodiment the gene is regulated in chronic neuropathic pain models). Ina more preferred embodiment, the genes encode for a P₁ or P₂ PurineReceptor, more preferably for a receptor of the P₂Y or P₂X subclass.Most preferred are the P₂X₃ or the P₂X₂ gene. Other examples ofpreferred genes include, but are not limited to the Vanilloid receptors1, voltage gated calcium channel (N-type), the tetrodotoxin-resistantsodium channel Nav1.8 (PN3/SNS), TRPM8, the Galanin R1 receptor or thegenes described in U.S. Patent Application 60/369,893 such as forinstance IL-24, IL-20Ralpha or IL-20Rbeta.

The pharmaceutical compositions disclosed herein useful for treatingand/or ameliorating chronic pain, including chronic neuropathic pain,are to be administered to a patient at therapeutically effective dosesto treat or ameliorate symptoms of such disorders. A therapeuticallyeffective dose refers to that amount of the compound sufficient toresult in amelioration of pain symptoms of chronic pain based on, forexample, use of the McGill pain score (Meizack, R. Pain (1975) September1(3):277-299).

Compositions and formulations for intrathecal administration may includesterile aqueous solutions which may also contain buffers, diluents andother suitable additives such as, but not limited to, penetrationenhancers, carrier compounds and other pharmaceutically acceptablecarriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. The pharmaceutical formulations of the present inventionmay be prepared according to conventional techniques well known in thepharmaceutical industry. Such techniques include the step of bringinginto association the active ingredients with the pharmaceuticalcarrier(s) or excipient(s). In general the formulations are prepared byuniformly and intimately bringing into association the activeingredients with carriers.

The compositions of the present invention may be formulated into any ofmany appropriate dosage forms. The compositions of the present inventionmay for instance be formulated as suspensions in aqueous or mixed media.Aqueous suspensions may further contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

A therapeutically effective dose refers to that amount of activeingredient, i.e. double-stranded RNA in accordance with the presentinvention, useful to treat and/or ameliorate the pathological effects ofchronic pain. Therapeutic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED50 (the dose therapeutically effective in 50% of thepopulation) and LD50 (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD50/ED50. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedata obtained from cell culture assays and animal studies is used informulating a range of dosage for human use. The dosage contained insuch compositions is preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors that may be taken intoaccount include the severity of the disease state, general health of thesubject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Guidance as toparticular dosages and methods of delivery is provided in the literatureand generally available to practitioners in the art.

Another aspect of the present invention provides the use of a doublestranded RNA for the preparation of a medicament for the treatment ofchronic pain. Preferably, the double stranded RNA inhibits purinereceptors P1 or P2 or Galanin R1 receptor or IL-24 or IL-20Ralpha orIL-20Rbeta or MMP7, more preferably Mob-5 or P2X3 or P2X2. Said chronicpain is preferably cancer pain or osteoarthritis pain, more preferablyhyperalgesia, most preferably allodynia.

The invention is further described by reference to the followingexamples. These examples are provided for illustration purposes and arenot intended to be limiting.

EXAMPLES

The following experiments are used to illustrate the invention.

Materials and Methods

Synthesis of Oligonucleotides Targeting P2X3 or MOB-5

Antisense Oligonucleotides (ASOs)

The ASOs against P₂X₃ and GAPDH are fully phosphorothioated 18-mers withnine nucleotides at the 3′-end modified with 2′-MOE groups and weresynthesized using phosphoramidite chemistry (Eur. Pat. Appl. EP 992506A2), HPLC-purified and characterized by electrospray mass spectrometryand capillary gel electrophoresis. For mismatch—containing controloligonucleotides, the approximate base composition of the matcholigonucleotides was maintained (Table 1).

TABLE 1 Sequences of ASOs used in in vitro experiments Target PositionRelation to GC SEQ gene in Cds^(a) other ASOs^(b) Sequence^(c) contentID 5037 rat P₂X₃ 785-802 5′-CsTsCsCsAsTsCsCsAsgscscs 61 1 gsasgstsgsa-3′5655 rat P₂X₃ 4 MM to 5037 5′-CsTsAsCsAsGsCsCsAstscscsg 61 2scsgstsgsa-3′ 5660 Rat 548-565 5′-GsGsCsCsAsTsCsCsAscsasgs 55 3 GAPDHtscststscst-3′ 5596 unrelated 5′-ccttaCsCsTsGsCsTsAsGsctggc-3′ 61 4^(a)cds = coding sequence ^(b)MM = mismatch(es)

Unless otherwise stated, internucleotidic linkages are phosphodiester,N=deoxyribonucleoside, n=2′-O-(2-methoxyethyl) ribonucleoside,c=2′-O-(2-methoxyethyl)5-methyl cytidine, t=2′-O-(2-methoxyethyl)5-methyl uridine, s=phosphorothioate.

TABLE 2 Sequences of ASOs used in in vivo experiments Target PositionRelation to GC SEQ gene in Cds^(a) other ASOs^(b) Sequence^(c) ID 6798rat P₂X₃ 785-802 5′-ctccaTCCAGCCGagtga-3′ 61 5 6799 rat P₂X₃4 MM to 5037 5′-ctacaGCCATCCGcgtga-3′ 61 6

All oligonucleotides are full phosphodiester 18mers; N isdeoxyribonucleoside, n=2′-O-(2-methoxyethyl) ribonucleoside,c=2′-O-(2-methoxyethyl) 5-methyl cytidine, t=2′-O-(2-methoxyethyl)5-methyl uridine.

Oligoribonucleotides (siRNA's)

Modified synthetic oligoribonucleotides and modified antisenseoligonucleotides described in this invention are prepared using standardphosphoramidite chemistry on ABI394 or Expedite/Moss Synthesizers(Applied Biosystems) for in vitro use and on OligoPilot II (AmershamPharmacia Biotech) for in vivo purpose. Phosphoramidites are dissolvedin acetonitrile at 0.05 M concentration (0.2M on Oligopilot II),coupling is made by activation of phosphoramidites by a 0.2 M solutionof benzimidazolium triflate in acetonitrile. Coupling times are usuallycomprised between 3-6 minutes. A first capping is made using standardcapping reagents. Sulfurization is made by using a 0.05 M solution ofN-ethyl, N-phenyl-5-amino-1,2,4-dithiazol-3-thione for two minutes(described in EP-A-0992506). Oxidation is made by a 0.1 M iodinesolution in THF/Pyridine/water (1:1:1) for two minutes. A second cappingis performed after oxidation or sulfurization. Oligonucleotide growingchains are detritylated for the next coupling by 2% dichloroacetic acidin dichloromethane or dichloroethane. After completion of the sequencesthe support-bound compounds are cleaved and deprotected as “Trityl-on”by a Methylamine solution (41% aqueous methylamine/33% ethanolicmethylamine 1:1 v/v) at 35° C. for 6 h for oligoribonucleotides and by a32% aqueous Ammonia solution at 55° C. for 16 h for antisenseoligonucleotides. Resulting suspensions are lyophilised to dryness. Foroligoribonucleotides, 2′-O-silyl groups are removed upon treatment with1M tetrabutylammonium fluoride 10 min at 50° C. and 6 h at 35° C. Theobtained crude solutions are directly purified by RP-HPLC. The purifieddetritylated compounds are analysed by Electrospray Mass spectrometryand Capillary Gel Electrophoresis and quantified by UV according totheir extinction coefficient at 260 nM. The oligoribonucleotides andantisense oligonucleotides directed against rat P2X3 and MOB-5 and theircontrols are shown in table 3 and 4, respectively.

TABLE 3 Oligoribonucleotides directed against rat P2X3. SEQ Name CommentSequence (5′- to 3′) ID NAS-8646 Sense strand UCACUCGGCUGGAUGGAGUtst 7NAS-8647 Antisense ACUCCAUCCAGCCGAGUGAasg 8 strand NAS-7556 Sense strandUCACUCGGCUGGAUGGAGUasa 9 NAS-7557 Antisense ACUCCAUCCAGCCGAGUGAasa 10strand NAS-7558 4 mismatch- UCACUGCGCUCGAUGCAGUasa 11 sense strandNAS-7559 4 mismatch ACUGCAUCGAGCGCAGUGAasa 12 antisense strand NAS-4882Sense strand UCACUCGGCUGGAUGGAGUdTdT 13 NAS-4883 AntisenseACUCCAUCCAGCCGAGUGAdTdT 14 strand NAS-4884 Sense strandGGCCUACCAAGUGAGGGACdTdT 15 NAS-4885 Antisense GUCCCUCACUUGGUAGGCCdTdT 16strand NAS-7126 Sense strand ACGGCAGCGUGCAGCUCGCCgsa 17 NAS-7127Antisense GGCGAGCUGCACGCUGCCGUcsc 18 strand NAS-10104 4 mismatch ofACUGCAUCGAGCGCAGUGAasg 19 NAS-8646 (antisense) NAS-10105 4 mismatch ofUCACUGCGCUCGAUGCAGUtst 20 NAS-8647 (sense)

RNAs NAS-8646 and NAS-8647, NAS-7556 and NAS-7557, NAS-4882 andNAS-4883, NAS-4884 and NAS-4885, NAS-7127 and 7126 as well as NAS-10104and NAS-10105 are annealed together to give the siRNA's. NAS-8646 andNAS-8647 as well as NAS-10104 and 10105 have 2′-MOE ribonucleotides atthe 3′-terminus of the oligoribonucleotide, and the sequence NAS 8647 isfully complementary to the target gene. NAS-7556 and NAS-7557 have2′-MOE-A ribonucleotides at the 3′-terminus of the oligoribonucleotides;NAS-4882 and NAS-4883 have 2-dT deoxyribonucleotides at the 3′-terminusof the oligoribonucleotide; NAS-4884 and NAS-4885 have 2-dTdeoxyribonucleotides at the 3′-terminus of the oligoribonucleotide. Theoligoribonucleotides NAS-7126 and 7127 target an unrelated gene. Unlessotherwise stated, internucleotidic linkages are phosphodiester,N=ribonucleoside, n=2′-O-(2-methoxyethyl) ribonucleoside,c=2′-O-(2-methoxyethyl)5-methyl cytidine, t=2′-O-(2-methoxyethyl)5-methyl uridine, dN=deoxyribonucleoside, s=phosphorothioate.

TABLE 4 Oligoribonucleotides and ASO's directed against rat MOB-5.Sequence SEQ Name Comment (5′- to 3′) ID NAS-11535 Antisense strandUUC AGC AGG CUG UGG 21 GCA AdGdG NAS-11536 Sense strandUUG CCC ACA GCC UGC 22 UGA AdTdT NAS-11537 4 mismatch-UUC CGA AGG CGG UGU 23 antisense strand GCA AdGdG NAS-11538 4 mismatchUUG CAC ACC GCC UUC 24 sense strand GGA AdTdT NAS-8154 ASOtca gcdA dGdGdC 25 dTdGdT dGgg caa NAS-7428 ASO aca gcTs CsTsCs 26GsGsCs Astc cga NAS-7429 ASO tca gcAs GsGsCs 27 TsGsTs Gsgg caa NAS-7443ASO tcc gaAs GsGsCs 28 GsGsTs Gstg caa NAS-4660 ASO GsGsCs CsAsTs CsCsAs29 csasgs tscsts tscst

RNAs NAS-11535 and NAS-11536, NAS-11537 and NAS-11538, are annealedtogether to give the siRNA's. The ASO NAS-4660 targets an unrelatedgene. Unless otherwise stated, internucleotidic linkages arephosphodiester, N=ribonucleoside, dN=deoxyribonucleoside,n=2′-O(methoxyethyl) ribonucleoside, s=phosphorothioate.

Generation of a CHO Cell Line Expressing Rat-P2X3 (rP₂X₃-CHO)

Chinese hamster ovary cells (CHO-K1, ATCC CCL61) were stably transfectedwith a complete rat P2X3 CDNA sequence in the vector pRK7 (kindlyobtained from John Wood; Chen, C. C et al. (1995). Nature 377,428-430.). To be able to select for transfected cells, vector wasco-electroporated in 10× excess with pMC1neo (Stratagene) containing aNeomycin resistance gene. Cells were cultured in Minimal EssentialMedium α (MEMα) supplemented with 10% (v/v) fetal bovine serum (FBS),2-mM glutamine, and 10.000 IU/500 ml Penicillin/Streptomycin.

Generation of a CHO Cell Line Expressing Rat-P2X3/P2X2 (rP₂X_(3/2)-CHO)

rP2X3 insert was obtained by PCR using as template RT from total rat DRGRNA and using the oligos listed below:

P2X3-Hind-F: (SEQ ID No 30) CGCAAGCTTGGCTGTGAGCAGTTTCTCAGTATGAACTTGP2X3-SacI-R: (SEQ ID No 31) CTTGAGCTCGGGAAGAGGCCCTAGTGACCAATAG

Note: the underlined sequence is a HindIII restriction enzyme site addedto the P2X3 complementary oligo. The SacI site in the reverse primer wasnot used for cloning.

The PCR product was amplified using Advantage-HF2 Polymerase withthermal cycling at 94° C. for 30 s, 62° C. for 60 s, and 68° C. for 180s for 6 cycles and 94° C. for 30 s, and 68° C. for 240 s for additional29 cycles The PCR fragment was cloned in pGEM T-Easy (Promega) andsequenced with T7 and M13 reverse primers. The clone had the samesequence as the Genbank cDNA with accession number X91167.

rP2X3 insert was cut out of pGEM T-Easy by digestion with NotI andsubcloned into pcDNA5/FRT linearized with NotI and dephosphorylated.

rP2X2 was obtained by digestion with BamHI and XhoI of a clone (seeabove). rP2X2 was subcloned into pcDNA5/FRT-Neo cut with the sameenzymes.

2.5 μg of each DNA was used to transfect CHO Flp-In cells containing twointegrated FRT sites using FuGENE 6 reagent. Cells transfected withrP2X2 have been selected with Geneticin 500 μg/ml, then transfected withrP2X3 and selected with Hygromycin-B 200 μg/ml to obtain the doubletransfectant.

Transfection of a Cell Line Expressing Rat-MOB-5

The cell line used was RBA (ATCC number 1747). It is rat skin derived,grows extremely tightly attached in a flattened out, skin-likemorphology, and naturally expresses ras and mob-5 (known to beras-downstream).

24 hours before transfection, 2×105 cells in a volume of 2 ml per wellwere plated into 6-well plates to yield 70-80% confluency. The day oftransfection, a 2 fold stock transfector-solution was prepared bydiluting Lipofectin® into serum-free OptiMEM (both GIBCO-BRL,Gaitherburg, Md.) (formula: 3 μl Lipofectin per 100 nM desired finaloligonucleotide concentration into 1 ml OptiMEM) and incubating for 15minutes at room temperature. This solution was then combined 1:1 with a2fold ASO-solution containing twice the desired final amount of ASO inOptiMEM. After incubating the transfection mixture for 15 minutes atroom temperature to form the transfection complex, 2 ml were added toeach of the previously aspirated well of cells. A Lipofectinreagent-only control and a normal cell control (untreated) were alsoincluded. After incubation for 4 hours at 37° C., 500 μl 50% FBS in MEMαwas added to each well to obtain a final FBS concentration of 10%. Thecultures were incubated at 37° C. in a humidified incubator with 5% CO2for 24 hours before mRNA was harvested.

Oligo, Average fg/ concentration 50 ng total RNA 7428, 400 nM 5.03E+027428, 200 nM 5.47E+02 7428, 100 nM 7.13E+02 7428, 50 nM 9.90E+02 7429,400 nM 1.73E+02 7429, 200 nM 1.97E+02 7429, 100 nM 4.63E+02 7429, 50 nM5.93E+02 7429, 12.5 nM 1.09E+03 7443, 400 nM 9.90E+02 7443, 200 nM1.21E+03 7443, 100 nM 9.73E+02 7443, 50 nM 6.77E+02 7443, 12.5 nM1.16E+03 Lipofectin 7.53E+02 Untreated 1.00E+03 4660, 400 nM 8.40E+024660, 200 nM 1.35E+03 4660, 100 nM 1.04E+03

Electroporation of Mammalian Cells

CHO-rP2X2/3 cells were transfected with 0.15; 0.3; 0.6 or 1.2 nmole ofASO or siRNA duplex using standard electrotransfection (10⁶ cells/125 ulin Biorad cuvette 0.4 cm, 250V, 0.3 μF, infinite resistance). Followingelectroporation, samples were immediately combined with 6 ml of theculture medium. In result, corresponding final concentration of ASO orsiRNA reagents were 10, 50, 100 or 200 nM. Cells were plated on uncoated96-well plates (Costar, Cat. #3904) and incubated at 37° C. for 24 h or48 h, followed by RNA or protein extraction, respectively.

Total RNA Isolation and Assay by Quantitative Real Time PCR (Q-PCR)

Total RNA was extracted and purified using RNeasy 96 kit (Qiagen).Primer pairs and FAM-labelled TaqMan probes for real time PCR weredesigned using the Primer Express v 2.0 program (ABI PRISM, PEBiosystems). For the Q-PCR reaction, 50 ng total RNA was mixed with 5′and 3′ primers (10 μM each), TaqMan probe (5 μM), MuLV reversetranscriptase (6.25 u, PE Biosystems), RNase Out RNase inhibitor (10 u,Life Technologies) and the components of the TaqMan PCR reagent kit(Eurogentec) in a total volume of 25 μl following the TaqMan PCR reagentkit protocol (Eurogentec). Reverse transcription and real time PCR wasperformed in a GeneAmp Sequence Detector 5700 (PE Biosystems) asfollows: 2 minutes reverse transcription at 50° C., 10 minutesdenaturation at 95° C. followed by 50 cycles of denaturation for 15 secat 95° C. and annealing and elongation for 1 min at 60° C. The relativequantitation of gene expression was calculated as described in the ABIPRISM 7700 user bulletin #2 (PE Biosystems).

Western Blotting

Cells grown in 6-well plates were washed with PBS and lysed with abuffer containing 141 mM NaCl, 5 mM KCl, 2.5 mM Tris pH7.4, 50 nMVa3VO4, 0.1% (v/v) Nonidet P-40 (100%), and 0.06 g protease inhibitorper 100 ml. Lysates were centrifuged for 10 min at 14000 rpm.Solubilized proteins in the supernatant were subjected toSDS-polyacrylamide gel electrophoresis through NuPAGE™ 4-12% Bis-TrisGels in a NOVEX™ Mini-Cell system, followed by transfer to PVDFmembranes (Millipore). The filters were blocked for 1 h with theblocking buffer contained in the ECF Western Blotting Kit (AmershamPharmacia Biotech), washed several times in 1× PBS, pH 7.4 with 0.05%Tween 20, and incubated for 1 h with the primary anti-P2X3-antibody(purchased from Neuromics) in a dilution 1:5000. With several washes inbetween, the filters were then incubated with the secondary antibody,tertiary antibody and ECF substrate from the ECF Western Blotting Kitfollowing the manufacturer's suggestions. A quantification of thevisualized bands was done with the software ImageQuant™ (MolecularDynamics).

FLIPR Assay—Generation and Analysis of FLIPR Data

FLIPR experiments were performed as follows. Briefly, cells were loadedwith fluo-4 AM in presence of 2.5 mM probenicid for 30-45 min, washedtwice with HBSS (Gibco)+20 mM HEPES, and transferred to the fluorescencereader (FLIPR, Molecular Devices). Drug plates were prepared at 5× thefinal concentration. Fluo-4 fluorescence was measured at a rate of 0.5Hz for 3 min. Agonists were applied after 20 points baseline detection.

FLIPR sequence files were analyzed using Igor Pro (Wavemetrics).Baseline was set as the average of 20 points before drug addition, peakwas detected as maximal signal in the 50 data points after drugaddition. Relative change of fluorescence (dF/F) was determined as(peak−baseline)/(baseline) values. These values were averaged, and forconcentration-response analysis further analyzed by fitting a sigmoidalhill equation to the data. Data are presented as mean+/− S.E.M. or EC50values as mean (95% confidence interval).

Animal Models of Chronic Pain

In vivo animal models of chronic neuropathic pain include the following:

Seltzer Model

In the Seltzer model (Seltzer et al. (1990) Pain 43: 205-218) rats areanaesthetised and a small incision made mid-way up one thigh (usuallythe left) to expose the sciatic nerve. The nerve is carefully cleared ofsurrounding connective tissues at a site near the trochanter just distalto the point at which the posterior biceps semitendinosus nerve branchesoff the common sciatic nerve. A 7-0 silk suture is inserted into thenerve with a ⅜ curved, reversed-cutting mini-needle, and tightly ligatedso that the dorsal ⅓ to ½ of the nerve thickness is held within theligature. The muscle and skin are closed with sutures and clips and thewound dusted with antibiotic powder. In sham animals the sciatic nerveis exposed but not ligated and the wound closed as in nonsham animals.

Chronic Constriction Injury (CCI) Model

In the CCI model (Bennett, G. J. and Xie, Y. K. Pain (1988) 33: 87-107)rats are anaesthetised and a small incision is made mid-way up one thigh(usually the left) to expose the sciatic nerve. The nerve is cleared ofsurrounding connective tissue and four ligatures of 4/0 chromic gut aretied loosely around the nerve with approximately 1 mm between each, sothat the ligatures just barely constrict the surface of the nerve. Thewound is closed with sutures and clips as described above. In shamanimals the sciatic nerve is exposed but not ligated and the woundclosed as in nonsham animals.

Chung Model

In contrast to the Seltzer and CCI models which involves damage toperipheral nerves, the Chung model involves ligation of the spinalnerve. (Kim, S. O. and Chung, J. M. Pain (1992): 50:355-363). In thismodel, rats are anesthetized and placed into a prone position and anincision is made to the left of the spine at the L4-S2 level. A deepdissection through the paraspinal muscles and separation of the musclesfrom the spinal processes at the L4-S2 level will reveal part of thesciatic nerve as it branches to form the L4, L5 and L6 spinal nerves.The L6 transverse process is carefully removed with a small rongeurenabling visualisation of these spinal nerves. The L5 spinal nerve isisolated and tightly ligated with 7-0 silk suture. The wound is closedwith a single muscle suture (6-0 silk) and one or two skin closure clipsand dusted with antibiotic powder. In sham animals the L5 nerve isexposed as before but not ligated and the wound closed as before.

Axotomy Model

The Axotomy model involves complete cut and ligation of the sciaticnerve. The nerve endings form neuromas but there is no behavioralcorrelate in this model as the nerve is not allowed to regenerate, andthe foot is permanently denervated. (Kingery and Vallin, Pain 38,321-32, 1989)

High Sciatic Lesion Model

In this model, the sciatic nerve is punctured in the region of the iliacarch. Although there is no overt damage to the nerve, local swellingproduces an increase in pressure on the nerve as it passes under theiliac arch. This model resembles conditions often seen in the clinic.

Chronic Inflammatory Pain Model

The Complete Freund's Adjuvant—induced mechanical hyperalgesia may beused as a model of chronic inflammatory pain (Stein, C. et al.Pharmacol. Biochem. Behav. (1988) 31:445-451). In this model, typicallya male Sprague-Dawley or Wistar rat (200-250 g) receives an intraplantarinjection of 25 μl complete Freund's adjuvant into one hind paw. Amarked inflammation occurs in this hind paw. Drugs are generallyadministered for evaluation of efficacy, 24 hours after the inflammatoryinsult, when mechanical hyperalgesia is considered fully established.

Behavioral Index

In all chronic pain models (inflammatory and neuropathic) mechanicalhyperalgesia is assessed by measuring paw withdrawal thresholds of bothhindpaws to an increasing pressure stimulus using an Analgesymeter(Ugo-Basile, Milan). Mechanical allodynia is assessed by measuringwithdrawal thresholds to non-noxious mechanical stimuli applied with vonFrey hairs to the plantar surface of both hindpaws. Thermal hyperalgesiais assessed by measuring withdrawal latencies to a noxious thermalstimulus applied to the underside of each hindpaw. With all models,mechanical hyperalgesia and allodynia and thermal hyperalgesia developwithin 1-3 days following surgery and persist for at least 50 days. Forthe assays described herein, drugs may be applied before and aftersurgery to assess their effect on the development of hyperalgesia,particularly approximately 14 days following surgery, to determine theirability to reverse established hyperalgesia.

The percentage reversal of hyperalgesia is calculated as follows:

${\%\mspace{14mu}{reversal}} = {\frac{{{postdose}\mspace{14mu}{threshold}} - {{predose}\mspace{14mu}{threshold}}}{{{naive}\mspace{14mu}{threshold}} - {{predose}\mspace{14mu}{threshold}}} \times 100}$

In the experiments disclosed herein, Wistar rats (male) are employed inthe chronic neuropathic pain models described above. Rats weighapproximately 120-140 grams at the time of surgery. All surgery isperformed under enflurane/O₂ inhalation anaesthesia.

In all cases the wound is closed after the procedure and the animalallowed to recover.

In all but the axotomy model, a marked mechanical and thermalhyperalgesia and allodynia develops in which there is a lowering of painthreshold and an enhanced reflex withdrawal response of the hind-paw totouch, pressure or thermal stimuli. After surgery the animals alsoexhibit characteristic changes to the affected paw. In the majority ofanimals the toes of the affected hind paw are held together and the footturned slightly to one side; in some rats the toes are also curledunder. The gait of the ligated rats varies, but limping is uncommon.Some rats are seen to raise the affected hind paw from the cage floorand to demonstrate an unusual rigid extension of the hind limb whenheld. The rats tend to be very sensitive to touch and may vocalise.Otherwise the general health and condition of the rats is good.

Treatment of Animal Models of Chronic Pain

Oligonucleotide reagents used in the animal models have been named asthe following:

ASO: NAS-6798 (SEQ. ID 5), MSO: NAS-6799 (SEQ. ID 6), P2X3 RNAi:NAS-8646 and NAS-8647 (SEQ. ID 7 and 8 respectively), P2X3 RNAimissense: NAS-10104 and NAS-10105 (SEQ. ID 19 and 20 respectively).

Intrathecal Delivery of siRNA.

dsRNA was administered intrathecally via an indwelling cannula in abuffer (100 mM KA 2 mM MgAc, 0.1749 g HEPES free acid (M=238.3), 0.2102g NaCl per 100 ml RNase free water; pH 7.63 at 20° C. with KOH). Ratswere anaesthetised and an incision made in the dorsal skin just lateralto the midline and approximately 10 mm caudal to the ventral iliacspines. A sterile catheter (polyethylene PE10 tubing) was inserted via aguide cannula (20 gauge needle) and advanced 3 cm cranially in theintrathecal space approximately to the L1 level. The catheter was thenconnected to an osmotic mini-pump (Alzet) delivering P2X3 receptor orMOB-5 receptor siRNA, missense siRNA or saline (1 μl/h, 7 days) whichwas inserted subcutaneously in the left or right flank. The incision wasclosed with wound clips and dusted with antibiotic powder. Experimentsdetermined 180 as well as 220 μg/day to have no signs of toxicity.Mechanical hyperalgesia was assessed on day 0, day 6 beforeadministration of α,β-methylene-ATP (Me-ATP) and 1 h post Me-ATP bymeasuring paw withdrawal thresholds of both hindpaws to an increasingpressure stimulus using an Analgesymeter (Ugo-Basile, Milan). Thecut-off was set at 250 g and the end-point taken as paw withdrawal,vocalisation or overt struggling. Each animal was tested only once, inrandom order. The statistical significance of mechanical hyperalgesiadata obtained from the different experimental animal groups was analysedusing ANOVA followed by Tukey's HSD test. 1.0 μmol (in 10 μl) of Me-ATPwas given intraplantar (ipl) to the contralateral hindpaw on the finalday of the experiment. 1.0 h post administration, paw withdrawalthresholds to mechanical hyperalgesia were measured.

Example 1 Transfection of siRNA's into Cell Lines Expressing Rat P₂X₃

treatment RNA [%] remaining SD OF 99 4 MM NAS-7558/7559 100 2NAS-7556/7557 18 2 NAS-8646/8647 11 3 NAS-4882/4883 11 6 NAS-4884/488521 4

Efficacy of P2X3 mRNA inhibition by variety of siRNA's delivered byOligofectamine-mediated transfection of CHO-rP2X3 cells. Q-PCR analysis.OF: control with Oligofectamine alone; mmNAS-7558/7559: control withmismatch; NAS-7556/7557: siRNA with optimised (as NAS-5037) sequence andnoncomplementary moe/ps overhangs; NAS-8646/8647: as NAS-7556/7557 butwith mRNA-complementary overhangs; NAS-4882/4883: siRNA with optimised(as NAS-5037) sequence and noncomplementary dTdT overhangs;NAS-4884/4885: siRNA with a sequence designed according to criteriadescribed in Harborth, J. et al. (2001). J. Cell Science 114: 4557-4565.

Example 2

Treatment % P2X3 protein remaining No treatment 90 Oligofectamine alone100 siRNA NAS-7556/7557 25 P2X3 mismatch NAS-7558/7559 100 SiRNAunrelated 100

P₂X₃ protein inhibition by the siRNA NAS-7556/7557, plotted as %. AfterSDS-PAGE, protein was blotted, immunodetected with anti-P₂X₃ antibody(Neuromics), blot bands were quantified with the software ImageQuant™,and expression levels were plotted as % against control siRNA.

Example 3

% mRNA SD Treatment ASO siRNA ASO siRNA untreated 100 100 2.4 2.4 MM 7177 3.0 5.6  10 nM 78 54 5.0 2.9  50 nM 71 34 2.4 4.1 100 nM 63 28 3.94.6 200 nM 44 22 5.2 8.5

Comparison of mRNA inhibition of P₂X₃ expression at 4 doses by the siRNANAS-8646/8647 and its antisense analogue NAS-5037. Electroporation ofthe CHO cell line expressing recombinant P2X3 and recombinant P2X2. mRNAwas measured with real-time quantitative PCR.

Example 4

% mRNA SD treatment ASO siRNA ASO siRNA untreated 100 100 4 4 MM 122 1093 3  10 nM 108 83 3 5  50 nM 88 67 4 2 100 nM 82 43 3 2 200 nM 67 31 2 3

FLIPR functional assay. Downregulation of functional response to 10 μMMe-ATP agonist by transfection of ASO-NAS-5037 and siRNA NAS-8646/8647into a CHO-r P2X2/P2X3 cell line at concentrations of 10, 50, 100, and200 nM 48 h prior to agonist treatment as compared to untreated controland to mismatch controls (MSO-5655 and siRNA NAS-7557/7558).

Example 5 Effect of P2X3 siRNA's on Hyperalgesia in Rats withAgonist-Induced Pain

Left paw Paw threshold (g) (agonist P2X3 ASO P2X3 RNAi P2X3 RNAiinjected) vehicle 180 μg 180 μg 220 μg Naïve day 0 101.7 ± 2.0 103.3 ±2.0 98.3 ± 4.1 101.7 ± 2.0  Predose day 6 100.0 ± 0.0 101.7 ± 2.0 101.7± 5.4  98.3 ± 2.0 1 h  58.3 ± 2.0  83.3 ± 2.0 75.0 ± 3.5 81.7 ± 7.4

The effect of P2X3 RNAi administered as an intrathecal infusion over 6days on naïve rats on mechanical allodynia. On day 6: ipl injection of 1μmol Me-ATP and measurement of Von Frey thresholds of these treatedrats. Vehicle: isotonic buffer, n=6/treatment group.

Example 6 Effect of P2X3 siRNA's on Allodynia in Rats withAgonist-Induced Pain

Von Frey threshold (g) Left paw (agonist P2X3 ASO P2X3 RNAi injected)vehicle 180 μg P2X3 RNAi 400 μg missense 400 μg Naïve day 0 15 ± 0  12.6± 1.5  14.2 ± 0.8   13 ± 1.3 Predose day 6 15 ± 0   14 ± 1.0 14.2 ± 0.8 14.1 ± 0.8  15 min post-dose 4.2 ± 0.9 4.4 ± 1.0 8.8 ± 1.3 3.3 ± 1.0 30min post-dose 4.5 ± 0.8   6 ± 0.9 9.1 ± 1.3 4.7 ± 1.0 60 min post-dose6.3 ± 0.8 6.4 ± 1.2 9.1 ± 1.3   5 ± 0.9

The effect of P2X3 RNAI administered as an intrathecal infusion over 6days on naïve rats on mechanical allodynia. On day 6: ipl injection of 1μmol Me-ATP and measurement of Von Frey thresholds of these treatedrats. Vehicle: isotonic buffer, n=6/treatment group.

Example 7 Effects of P2X3 siRNA on Mechanical Hyperalgesia in Rats withNeuropathic Pain (Seltzer Model)

Paw withdrawal thresholds (g) Neuropathic P2X3 RNAi Naive missense P2X3RNAi P2X3 ASO Day vehicle vehicle 400 μg 400 μg 180 μg  0  102.5 ± 1.33102.5 ± 1.33 104.375 ± 1.13  101.25 ± 0.81  102.5 ± 0.94 — — — — — — 11103.75 ± 1.57 58.12 ± 1.62   60 ± 1.64   60 ± 2.11  57.5 ± 1.63 12101.87 ± 1.61 61.25 ± 1.57 61.25 ± 1.83   80 ± 0.94   75 ± 0.94 13 102.5 ± 1.33  62.5 ± 2.11   60 ± 1.33 81.87 ± 2.09 74.37 ± 2.40 14103.12 ± 0.91 61.87 ± 1.88 64.37 ± 2.74 81.87 ± 1.87 76.25 ± 1.25 15103.75 ± 0.81 63.75 ± 1.25 64.37 ± 4.27 81.87 ± 1.61 75.62 ± 1.13 16101.875 ± 1.62  64.37 ± 1.13   65 ± 4.00 83.12 ± 0.91 73.12 ± 2.10 17 102.5 ± 1.64 61.87 ± 2.09 63.12 ± 4.21 79.37 ± 1.75 71.25 ± 2.63   17.5101.25 ± 1.56 63.75 ± 1.25  62.5 ± 3.13 81.25 ± 1.25 71.87 ± 2.30

Four groups of rats were ligated on the left hind limb on day 0 and baseline mechanical hyperalgesia was measured daily. An additional unligatedgroup (naïve) was set up as control. Rats were cannulated on day 11 andinfused with vehicle, RNAi, RNAi missense or ASO for a further 6 days.Paw withdrawal thresholds (left paw) were measured daily. Vehicle:isotonic buffer, n=8/treatment group.

Example 8 Effects of P2X3 siRNA on Mechanical Allodynia in Rats withNeuropathic Pain (Seltzer Model)

Von Frey thresholds (g) Neuropathic P2X3 RNAi Naive missense P2X3 RNAiP2X3 ASO Day vehicle vehicle 400 μg 400 μg 180 μg  0 13.75 ± 0.81 13.75± 0.81  13.75 ± 0.81  13.75 ± 0.81  15 ± 0  — — — — — — 11 14.37 ± 0.633.87 ± 1.17 3.25 ± 0.84 4.37 ± 1.28 4.125 ± 1.00  12 13.75 ± 0.81 4.37 ±1.28 3.25 ± 1.04  6.5 ± 1.05  4.5 ± 0.98 13 13.12 ± 0.91 4.75 ± 1.193.25 ± 0.99   8 ± 1.13 4.25 ± 1.03 14 14.37 ± 0.62 5.25 ± 0.92 3.25 ±0.99 7.25 ± 1.19 4.25 ± 0.96 15 14.37 ± 0.62 4.75 ± 1.25 3.25 ± 0.757.75 ± 1.10 4.25 ± 0.96 16 15 ± 0  4.5 ± 0.98   4 ± 0.75   7 ± 1.06  3.5± 0.73 17 14.37 ± 0.62 5 ± 1   4 ± 0.75 7 ± 1 4.25 ± 0.95

Four groups of rats were ligated on the left hind limb on day 0 and baseline mechanical allodynia was measured daily. An additional unligatedgroup (naïve) was set up as control.

Rats were cannulated on day 11 and infused with vehicle, RNAi, RNAimissense or ASO for a further 6 days. Von Frey thresholds on the leftpaw were measured daily. Vehicle: isotonic buffer, n=8/treatment group.

Example 9 Effects of MOB-5 siRNA on Mechanical Hyperalgesia in Rats withNeuropathic Pain (Seltzer Model)

Paw withdrawal thresholds (g) Neuropathic MOB-5 RNAi Naive missenseMOB-5 RNAi Day vehicle vehicle 400 μg 400 μg  0 101.4 ± 2.4 103.1 ± 1.399.4 ± 1.1 100 ± 1.9  — 10 101.4 ± 1.8 60.6 ± 1.5 62.5 ± 1.3  60 ± 1.611 102.1 ± 1.5 61.9 ± 1.3 66.2 ± 1.6 67.5 ± 2.1  12 102.1 ± 2.1 62.5 ±0.9 65.5 ± 1.1  75 ± 2.1 13   105 ± 2.2 63.7 ± 1.5 66.9 ± 1.6 81.2 ±2.9  14 102.1 ± 2.1 64.4 ± 1.5 62.5 ± 2.3 78.7 ± 4.2  15 102.1 ± 1.8 62.5 ± 0.94 61.9 ± 2.3 74.4 ± 2.7  16 102.1 ± 1.5  62.5 ± 0.94 58.7 ±2.3  75 ± 2.3

Four groups of rats were ligated on the left hind limb on day 0 and baseline mechanical hyperalgesia was measured. An additional unligated group(naïve) was set up as control. Rats were cannulated on day 10 andinfused with vehicle, RNAi or missense for a further 6 days. Pawwithdrawal thresholds (left paw) were measured daily. Vehicle: isotonicbuffer, n=8/treatment group. The right paw for each group were alsomeasured but showed no difference in paw withdrawal threshold to naïveanimals.

Example 10 Effects of MOB-5 siRNA on Mechanical Allodynia in Rats withNeuropathic Pain (Seltzer Model)

Von Frey thresholds (g) Neuropathic MOB-5 RNAi Naive missense MOB-5 RNAiDay vehicle vehicle 400 μg 400 μg  0 14.3 ± 0.7  12 ± 1.2 11.9 ± 0.9 12.9 ± 1.0  — 10 15 ± 0 5.9 ± 1.5   3 ± 0.6 6.1 ± 1.5 11 14.3 ± 0.7 5.5± 1.1 2.7 ± 0.5 5.7 ± 0.9 12 14.3 ± 0.7 4.2 ± 1.2 2.5 ± 0.5 6.2 ± 0.8 1314.3 ± 0.7 4.2 ± 1.2 2.7 ± 0.5 5.7 ± 0.8 14 15 ± 0 4.0 ± 1.0 2.5 ± 0.36.7 ± 0.8 15 12.1 ± 1.0 3.7 ± 0.7 2.5 ± 0.3 6.5 ± 0.7 16 12.1 ± 1.0 4.2± 0.7 3.2 ± 0.6 6.7 ± 0.7

Four groups of rats were ligated on the left hind limb on day 0 and baseline mechanical allodynia was measured. An additional unligated group(naïve) was set up as control. Rats were cannulated on day 10 andinfused with vehicle, RNAi or missense for a further 6 days. Von Freythresholds on the left paw were measured daily. Vehicle: isotonicbuffer, n=8/treatment group. The right paw for each group were alsomeasured but showed no difference in paw withdrawal threshold to naïveanimals.

The invention claimed is:
 1. A method to treat or ameliorate pain in asubject in need thereof, the method comprising the step of intrathecalinjection of an effective amount of a siRNA into the subject, whereinsaid siRNA inhibits the expression of the P₂X₃ gene by an RNAinterference mechanism and wherein more than 180 μg of siRNA per 120-140grams of body weight per day are intrathecally injected.
 2. A methodaccording to claim 1 wherein said pain is chronic neuropathic pain.
 3. Amethod according to claim 2 wherein said chronic pain is selected fromthe group consisting of cancer pain, osteoarthritis pain, allodynia andhyperalgesia.
 4. A method according to claim 1 wherein the subject inneed is a human.
 5. A method according to claim 1 wherein the subject inneed is a rat.
 6. A method according to claim 1 wherein at least 200 μgof siRNA per 120-140 grams of body weight per day are intrathecallyinjected.
 7. A method according to claim 1 wherein the siRNA comprises adouble-stranded region of 15 to 25 nt.
 8. A method according to claim 1wherein the siRNA comprises a 3′ overhang on the antisense or the sensestrand, or both strands, of at least one nucleotide.
 9. A methodaccording to claim 8 wherein the overhang contains at least one modifiednucleotide.
 10. A method according to claim 8 wherein the overhangcomprises at least one 2′-MOE modified nucleotide.
 11. A methodaccording to claim 8 wherein the overhang comprises 4 Uracils.
 12. Amethod according to claim 1 wherein the siRNA comprises at least onephosphorothioate linkage.
 13. A method to treat or ameliorate pain in asubject in need thereof, wherein the method comprises the step ofintrathecal injection into the subject of an effective amount of acomposition comprising a siRNA targeting the gene P₂X₃, wherein thesiRNA targets a P₂X₃ gene sequence corresponding to that of adouble-stranded RNA having any of the sequences of SEQ ID NOs: 7-10 or13-16, wherein the composition inhibits the expression of the P₂X₃ geneby an RNA interference mechanism.
 14. The method according to claim 13,wherein said pain is chronic neuropathic pain.
 15. The method accordingto claim 14, wherein said chronic pain is selected from the groupconsisting of cancer pain, osteoarthritis pain, allodynia andhyperalgesia.
 16. A method to treat or ameliorate pain in a subject inneed thereof, the method comprising the step of intrathecal injection ofan effective amount of a siRNA, wherein said siRNA inhibits theexpression of the P₂X₃ gene by an RNA interference mechanism, andwherein the dosage of siRNA is more than 180 μg of siRNA per 120-140grams of body weight per day.
 17. The method according to claim 16,wherein said pain is chronic neuropathic pain.
 18. The method accordingto claim 17, wherein said chronic pain is selected from the groupconsisting of cancer pain, osteoarthritis pain, allodynia andhyperalgesia.
 19. The method according to claim 16, wherein the subjectin need is a human.
 20. The method according to claim 16, wherein thesiRNA comprises a double-stranded region of 15 to 25 nt.
 21. The methodaccording to claim 16, wherein the siRNA comprises a 3′ overhang on theantisense or the sense strand, or both strands, of at least onenucleotide.
 22. The method according to claim 21, wherein the overhangcontains at least one modified nucleotide.
 23. The method according toclaim 21, wherein the overhang comprises at least one 2′-MOE modifiednucleotide.
 24. The method according to claim 21, wherein the overhangcomprises 4 Uracils.
 25. The method according to claim 16, wherein thesiRNA comprises at least one phosphorothioate linkage.